Peptidic antagonists of class iii semaphorins/neuropilins complexes

ABSTRACT

The present invention concern a peptidic antagonist of class III semaphorins/neuropilins complexes comprising an amino acid sequence, which is derived from the transmembrane domain of a protein selected in the group consisting of neuropilin-1, neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrin Beta 1 and integrin beta 2, and including at least a GxxxG motif, eventually fused to an heterologous sequence; a nucleic acid encoding for said peptidic antagonist, a pharmaceutical composition comprising such a peptidic antagonist or a nucleic acid encoding thereof and uses thereof.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/993,509, filed Oct. 10, 2008, which is a national stage ofInternational Application No. PCT/IB2006/002334, filed Jun. 28, 2006,which claims priority to European Application No. 05291392.8 filed Jun.28, 2005. The contents of each of the foregoing are incorporated hereinby reference.

The present invention relates to peptidic antagonists of class IIIsemaphorins/neuropilins complexes, and uses thereof.

BACKGROUND OF THE INVENTION

Next to its structural and trophic roles, the extra cellular matrix(ECM) defines an ideal environment for cell to cell communication anddetermines all the cellular behaviors, including proliferation,migration, differentiation or apoptosis. The molecular mechanismscontrolling these processes are getting better understood. In thenervous system, 3 major families of diffusible or transmembrane signals(netrins, semaphorins and ephrins) ensure these functions duringembryonic development (TESSIER-LAVIGNE and GOODMAN, Science, vol. 274,p: 1123-33, 1996). Among them, the semaphorins define a family of morethan 25 members subdivided into 8 classes according to their structuralspecificities (KOLODKIN et al., Cell, vol. 75, p: 1389-99, 1993) andwhich can be classified as either secreted or transmembrane semaphorins.The secreted ones are class II (invertebrates), III (vertebrates), and V(viral), whereas the other classes (I, IV and VI-VIII) aretransmembrane.

Over the past five years, several studies were designed to elucidate thetransduction pathways allowing the signaling of the diverse functions ofsemaphorins ranging from axon guidance, cell migration, celldifferentiation to apoptosis in both physiological and pathologicalconditions. The current view considers that this functional diversity isdue to the formation of a receptor complex, highly dynamic, modulatingsignal integration by selective recruitment and activation of multipleintracellular pathways leading to actin cytoskeleton remodeling(CASTELLANI and ROUGON, Curr. Opin. Neurobiol., vol. 12, p: 532-41,2002).

All of them have a common domain called the “sema” domain of nearly 500amino acids with 12-16 cysteine residues, which confers the bindingspecificity of each semaphorin (RAPER, Curr. Opin. Neurobiol., vol. 10,p: 88-94, 2000). Among these different semaphorins, class IIIsemaphorins induce the collapse of neuronal growth cones, which is whythey were initially named collapsins (LUO et al., Cell, vol. 75, p:217-227, 1993). Sema 3A, the molecule that gives the rest of the familyits name, is the most extensively studied, and in all cases it has beendescribed as a repellent factor for axons, from sensory neurons andspinal motoneurons to pyramidal neurons of the cortex (MUELLER, Annu.Rev. Neurosci., vol. 22, p: 351-388, 1999). Strikingly, this semaphorincan exert two different effects in the same cell. This has beendemonstrated in cortical neurons in which Sema3A acts as a repellentfactor for axons and is a chemoattractant for the dendrites (POLLEUX etal., Nature, vol. 404, p: 567-73, 2000; BAGNARD et al., Development,vol. 125(24), p: 5043-53, 1998). In order to explain this phenomenon, itis necessary to consider the existence of a mechanism ensuring adifferential transduction in the two cellular poles. More than aprinciple of differential transduction, it is necessary to understandthe mechanisms controlling the molecular hierarchy and to elucidate theformation of supra-molecular structures ensuring the diversity of thecellular behaviors in response to environmental changes.

Hence, recent works demonstrated the role of the two known members ofthe neuropilin family, neuropilin-1 (NRP1) and neuropilin-2 (NRP2), asthe ligand binding sub units of the receptor complex involved in thetransduction cascade of class III semaphorins (for review see Bagnard D.(Editor) Neuropilin: from nervous system to vascular and tumor biology.Landes Bioscience-Kluwer Academic/Plenum Publishers Hardbound, ISBN0-306-47416-6, Advance in Experimental Medicine and Biology Vol. 515, p:140, 2002). NRP1 and NRP2 are single spanning transmembrane proteinswith an (i) extracellular part, which is important for dimerization(RENZI et al., J. Neurosci., vol. 19, p: 7870-7880, 1999), atransmembrane segment, and a short cytoplasmic domain of about 40 aminoacids.

Interestingly, NRP1 and NRP2 possess a short intracellular domainwithout transduction capacity. A molecular explanation for thisobservation was given when it was found that neuropilins form complexeswith receptors belonging to the plexin family, and that the plexin isthe transducing element in neuropilin/plexin complex (RHOM et al., Mech.Dev., vol. 93, p: 95-104, 2000; TAMAGONE et al., Cell, vol. 99, p:71-80, 1999). Finally, signal transduction by class III semaphorinsdepends upon complex formation between neuropilins with the plexins.

Nevertheless, complexes with plexins are not the only types of complexesformed by neuropilins.

It was found that neuropilins can also form stable complexes with theadhesion molecules L1-CAM and Nr-CAM (CASTELLANI et al., Neuron, vol.27, p: 237-249, 2000) and mutations in the extracellular domain of L1 orthe complete absence of L1 in gene-targeted mice result in thedisruption of Sema 3A signaling leading to guidance errors.

Tyrosine kinase receptors may, therefore, also play a role inneuropilin-associated signaling. Thus, it has been observed that themigration of DEV neuroectodermal progenitor cells is repulsed by Sema3A, and the presence of both NRP1 and VEGFR-1 is required for therepulsion (Bagnard et al., J Neurosci., vol. 21, p: 3332-41, 2001). Thisinteraction explain the inhibition of sprout formation by VEGF in an invitro model of angiogenesis with Sema 3A (MIAO et al., J. Cell. Biol.,vol. 146, p: 233-242, 1999). It has also been found that neuropilinsform complexes with VEGFR-2 (SOKER et al., Cell, vol. 92, p: 735-45,1998) and MET (WINBERG et al., Neuron, vol. 32, p: 53-62, 2001).

Recently, it has also been shown that neuropilins form complexes withintegrins, and said complexes are able to promote axon outgrowth(PASTERKAMP et al., Nature, vol. 424, p: 398-405, 2003).

Consequently, the above studies contribute to identify class IIIsemaphorins/neuropilins complexes as a potential target forneurodegenerative conditions and cancer as recently evidence (for areview see GUTTMANN-RAVIV et al., Cancer Letter, 2006; CHEDOTAL et al.,Cell Death and Differentiation, 2005). In this context, agents thatinterfere with the complex formation would clearly have therapeuticpotential and/or be useful biological tools.

In this way, GARETH et al. (Journal of Neurochemistry, vol. 92, p:1180-1190, 2005) have used an algorithm in order to design a peptideantagonist of Sema 3A/NRP1 complex. The authors have identifiedantagonist peptides in the Sema 3A Ig domain, which is implicated inSema 3A/NRP1 dimerization, and a NRP1 MAM domain, which mediates thelateral dimerization of the receptor but not the ligand binding. Theidentified antagonist peptides are able to effectively inhibit thegrowth cone collapse response stimulated by Sema 3A. Nevertheless, theseantagonists, which are not located in the transmembrane domain, have anIC50 of more than 1 μM, said concentration being too important to enablethe use of such an antagonist in therapy.

So, there is a recognized and permanent need in the art for newantagonists of class III semaphorins/neuropilins complexes, which can beused in therapies.

DESCRIPTION OF THE INVENTION

The purpose of the present invention is to fulfil this need by providingnew antagonists having a greater activity.

Unexpectedly, the inventors have demonstrated that a peptidecorresponding to the transmembrane domain of NRP1 can inhibit thecortical axons collapses induced by Sema 3A with an IC50 of less than10⁻¹¹ M. This peptide includes two consecutive GxxxG dimerization motifs(where x represents any amino acid), said GxxxG motif was firstlyidentified in Glycophorin A (SENES et al., J. Mol. Biol., vol. 296, p:921-36, 2000). Such a motif has also been shown as operative in thedimerization of TM segments of erbB receptors (MENDROLA et al., J. Biol.Chem., vol. 277, p: 4704-12, 2002). In general any motif composed ofsmall amino acid-XXX-small amino acid (where the definition of a smallamino acid is well known from one of skill in the art) possessesequivalent properties.

The NRP1 double GxxxG motif is highly conserved and presents a stronghomology to the one of NRP2 (FIG. 1). Finally, such motifs are found inthe transmembrane domains of multiple partners of neuropilins includingmembers of the Plexin family, Nr- and L1-CAM, and integrins. Altogether,the results obtained by the inventors suggest that the transmembranedomains of these proteins have a key role in the formation andmodulation of the complexes ensuring semaphorin signaling.

Consequently, in one aspect the present invention relates to a peptidicantagonist of class III semaphorins/neuropilins complexes comprising anamino acid sequence, which is derived from the transmembrane domain of aprotein selected in the group consisting of neuropilin-1, neuropilin-2,plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrinbeta 1 and integrin beta 2, and including at least one GxxxG motif,optionally fused to an heterologous sequence.

As used herein an “heterologous sequence” relates to any amino acidsequence which is not derived from neuropilin-1, neuropilin-2,plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrinbeta 1 or integrin beta 2. This heterologous sequence can for exampleallows a specific cellular location or a better purification yield (e.g.His tag) of the peptidic antagonist of the invention.

As used herein the term “peptidic antagonist of class IIIsemaphorins/neuropilins complexes” relates to a synthetic or recombinantpolypeptide, which interferes with said complexes formation and finallythe signal transduction of such complexes. Consequently, the peptidicantagonists of the invention does not include the complete neuropilin-1,neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM,L1-CAM, integrin beta 1, and integrin beta 2 proteins.

As used herein “a transmembrane domain” corresponds to peptidic domaintraversing the cell's membrane. Said domain is hydrophobic and has anα-helical structure. One of skill in the art can simply identify suchdomains in said proteins according to its general knowledge. As anexample, the hydrophobicity of a proteic domain can be determined by theKyte & Doolittle method, and the potentiality of a proteic domain toform an-helical structure can be determined by the Chou & Fasman method.Such methods are notably available at the Expasy Bioinformatics ResourcePortal.

The amino acid sequence of the transmembrane domain of neuropilin-1,neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM,L1-CAM, integrin beta 1 and integrin beta 2 are well conserved and canbe simply identified from the complete amino acid sequence of theprotein, which are well known from one of skill in the art. As anexample, one can cite the neuropilin-1 amino acid sequence from Musmusculus (P97333), Homo sapiens (014786), Rattus norvegicus (Q9QWJ9),Zebrafish (Q8QFX6) and Gallus gallus (P79795), the neuropilin-2 aminoacid sequence from Homo sapiens (060462), Mus musculus (035375), Rattusnorvegicus (NP 110496) and Gallus gallus (NP_(—)989615), the plexin A-1amino acid sequence from Homo sapiens (NP_(—)115618, Q9UIW2) and Musmusculus (NP_(—)032907, P70206), the plexin A-2 amino acid sequence fromHomo sapiens (CAI40198, Q5JRL6) and Mus musculus (NP_(—)032908, P70207),the plexin A3 amino acid sequence from Homo sapiens (NP_(—)032907,P51805), Xenopus tropicalis (CAI40198) and Mus musculus (NP_(—)032909,P70208), the plexin A4 amino acid sequence from Mus musculus(NP_(—)786926, Q80UG2), Homo sapiens (Q9HCM2) and Danio rerio(NP_(—)001004495), the Nr-CAM amino acid sequence from Mus musculus(Q810U4), Rattus norvegicus (P97686), Homo sapiens (Q92823) and Gallusgallus (P35331), the L1-CAM amino acid sequence from Homo sapiens(P32004), Takifugu rubripes (Q98902), Mus musculus (P11627) and Rattusnorvegicus (Q05695), the integrin beta 1 amino acid sequence from Musmusculus (P09055), Homo sapiens (P05556), Felis catus (P53713), Rattusnorvegicus (P49134), Xenopus laevis (P12606) and Gallus gallus (P07228),and the integrin beta 2 amino acid sequence from Mus musculus (P11835),Homo sapiens (P05107), Sus scrofa (P53714), Bos taurus (P32592) andSigmodon hispidus (AAL38579).

The FIG. 1 shows the transmembrane domains of mouse neuropilin-1 (SEQ IDNO. 1: ILITIIAMSALGVLLGAVCGVVL), neuropilin-2 (SEQ ID NO. 2:ILITIIAMSSLGVLLGATCAGLLLY), plexin A1 (SEQ ID NO. 3:LLTLPAIVGIGGGGGLLLLVIVAVLIA), plexin A2 (SEQ ID NO. 4:LLTLPAIISIAAGGSLLLIIVIIVLIAY), plexin A3 (SEQ ID NO. 5:LTLPAMVGLAAGGGLLLLAITVVLVAY), plexin A4 (SEQ ID NO. 6:LSLPAIVSIAVAGGLLIIFIVAVLIA), Nr-CAM (SEQ ID NO. 7:GWFIGLMCAVALLILILLIVCF), L1-CAM (SEQ ID NO. 8: GWFIAFVSAIILLLLILLILCFI),integrin beta 1 (SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) and integrinbeta (SEQ ID NO. 10: VAAIVGGTVVGVVLIGVLLLVIW). The term “GxxxG motif”relates to the motif as identified in SENES et al. (above mentioned,2000), which is shown in FIG. 1 (underlined). Potential “GxxxG” motifare also shown (dotted line).

The conservation of these transmembrane domains clearly stands out fromthe FIG. 2, which shows the same transmembrane domains for humanneuropilin-1 (SEQ ID NO. 1: ILITIIAMSALGVLLGAVCGVVL), neuropilin-2 (SEQID NO. 2: ILITIIAMSSLGVLLGATCAGLLLY), plexin A1 (SEQ ID NO. 3:LLTLPAIVGIGGGGGLLLLVIVAVLIA), plexin A2 (SEQ ID NO. 11:LLTLPAIVSIAAGGSLLLIIVIIVLIAY), plexin A3 (SEQ ID NO. 12:LTLPAMMGLAAGGGLLLLAITAVLVA), plexin A4 (SEQ ID NO. 6:LSLPAIVSIAVAGGLLIIFIVAVLIA), Nr-CAM (SEQ ID NO. 7:GWFIGLMCAVALLILILLIVCFI), L1-CAM (SEQ ID NO. 13: GWFIGFVSAIILLLLVLLIL),integrin beta 1 (SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) and integrinbeta 2 (SEQ ID NO. 14: IAAIVGGTVAGIVLIGILLLVIW).

As another example of this conservation, one can cite the neuropilin-1transmembrane domain from Gallus gallus (SEQ ID NO. 14:ILITIIAMSALGVLLGAICGVVL), and from Zebrafish (SEQ ID NO. 15:ILITIIAMSALGVFLGAICGVVL), and the neuropilin-2 transmembrane domain fromGallus gallus (SEQ ID NO. 16: ILVTIIAMSSLGVLLGATCAGLLLY), which share anidentity of more than 90% with the human neuropilin-1 and neuropilin-2transmembrane domain respectively.

According to a preferred embodiment, the invention relates to a peptidicantagonist of class III semaphorins/neuropilins complexes comprising anamino acid sequence, which is derived from the transmembrane domain of aprotein selected in the group consisting of human neuropilin-1 (SEQ IDNO. 1: ILITIIAMSALGVLLGAVCGVVL), neuropilin-2 (SEQ ID NO. 2:ILITIIAMSSLGVLLGATCAGLLLY), plexin A1 (SEQ ID NO. 3:LLTLPAIVGIGGGGGLLLLVIVAVLIA), plexin A2 (SEQ ID NO. 11:LLTLPAIVSIAAGGSLLLIIVIIVLIAY), plexin A3 (SEQ ID NO. 12:LTLPAMMGLAAGGGLLLLAITAVLVA), plexin A4 (SEQ ID NO. 6:LSLPAIVSIAVAGGLLIIFIVAVLIA), Nr-CAM (SEQ ID NO. 7:GWFIGLMCAVALLILILLIVCFI), L1-CAM (SEQ ID NO. 13: GWFIGFVSAIILLLLVLLIL),integrin beta 1 (SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) and integrinbeta 2 (SEQ ID NO. 14: IAAIVGGTVAGIVLIGILLLVIW), and including at leastone GxxxG motif, optionally fused to an heterologous sequence.

According to another preferred embodiment, said peptidic antagonist ofclass III semaphorins/neuropilins complexes comprises an amino acidsequence, which is derived from the human neuropilin-1 (SEQ ID NO. 1,ILITIIAMSALGVLLGAVCGVVL) or neuropilin-2 (SEQ ID NO. 2,ILITIIAMSSLGVLLGATCAGLLLY) transmembrane domain, optionally fused to anheterologous sequence.

According to still another preferred embodiment, said peptidicantagonist of class III semaphorins/neuropilins complexes comprises anamino acid sequence, which is derived from the transmembrane domain of aprotein including at least two GxxxG motifs, preferably at least twoconsecutive GxxxG motifs, and selected in the group consisting of humanneuropilin-1 (SEQ ID NO. 1, ILITIIAMSALGVLLGAVCGVVL), integrin beta 1(SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) and integrin beta 2 (SEQ ID NO.14: IAAIVGGTVAGIVLIGILLLVIW) transmembrane domain, optionally fused toan heterologous sequence.

Preferably, the peptidic antagonist of the invention comprises an aminoacid sequence derived from the human neuropilin-1 transmembrane domain(SEQ ID NO. 1, ILITIIAMSALGVLLGAVCGVVL).

Advantageously, said amino acid sequence which is derived from atransmembrane domain of one of the proteins above described is more than10 amino acids length, preferably more than 14 amino acids length, as anexample more than 18 amino acids length, and more preferably more than22 amino acids.

Advantageously, said amino acid sequence which is derived from one ofthe proteins selected in the group consisting of neuropilin-1,neuropilin-2, plexin-A1, plexin-A2, plexin-A3, plexin-A4, Nr-CAM,L1-CAM, integrin beta 1 and integrin beta 2 is less than 150 amino acidsin length, preferably less than 100 amino acids in length, morepreferably less than 50 amino acids in length.

According to a preferred embodiment, the peptidic antagonists derivedfrom neuropilin-1 and neuropilin-2 do not include their extracellulardomains associated with class III semaphorins dimerization. Thesedomains are well known from one of skill in the art and are described inNEUFELD et al. (TCM, vol. 12(1), p: 13-19, 2002) and in BAGNARD (2002,above mentioned). For example, these domains include the a (CUB domain,also called a1 and a2 domains ensuring semaphorin binding), b (homologuedomain to coagulation factor V/VIII; subdivided into b1 and b2 domains,b1 being involved in the binding of VEGF isoforms), c (MAM domain,involved in the dimerization of NRP1) domains of NRP1 and NRP2.

Advantageously, said peptidic antagonist consists of an amino acidsequence selected in the group consisting of plexin A1 (SEQ ID NO. 3:LLTLPAIVGIGGGGGLLLLVIVAVLIA), plexin A2 (SEQ ID NO. 11:LLTLPAIVSIAAGGSLLLIIVIIVLIAY), plexin A3 (SEQ ID NO. 12:LTLPAMMGLAAGGGLLLLAITAVLVA), plexin A4 (SEQ ID NO. 6:LSLPAIVSIAVAGGLLIIFIVAVLIA), Nr-CAM (SEQ ID NO. 7:GWFIGLMCAVALLILILLIVCFI), L1-CAM (SEQ ID NO. 13: GWFIGFVSAIILLLLVLLIL),integrin beta 1 (SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) and integrinbeta (SEQ ID NO. 14: IAAIVGGTVAGIVLIGILLLVIW) transmembrane domains, orderivatives thereof, optionally fused to an heterologous sequence.

According to a second preferred embodiment, the peptidic antagonistsderived from plexin-A1, plexin-A2, plexin-A3, plexin-A4, integrin beta1,integrin beta2, Nr-CAM and L1-CAM do not include their intracellulardomains associated with signal transducing pathways. These domains arewell known from one of skill in the art and are described in BAGNARD(above mentioned, 2002) and include for example but not exclusively theSex-Plexin domain, the PH1A and PH2A domains or the PRB (Plexin Racbinding domain) domain.

Advantageously, said peptidic antagonist consists of an amino acidsequence selected in the group consisting of neuropilin-1 (SEQ ID NO. 1:ILITIIAMSALGVLLGAVCGVVL) and neuropilin-2 (SEQ ID NO. 2:ILITIIAMSSLGVLLGATCAGLLLY) transmembrane domains, or derivativesthereof, optionally fused to an heterologous sequence. Preferably, saidpeptidic antagonist consists of neuropilin-1 (SEQ ID NO. 1:ILITIIAMSALGVLLGAVCGVVL) transmembrane domain, or derivatives thereof,optionally fused to an heterologous sequence.

An amino acid sequence “derived from” or a “derivative of” thetransmembrane domain of human neuropilin-1 (SEQ ID NO. 1:ILITIIAMSALGVLLGAVCGVVL), neuropilin-2 (SEQ ID NO. 2:ILITIIAMSSLGVLLGATCAGLLLY), plexin A1 (SEQ ID NO. 3:LLTLPAIVGIGGGGGLLLLVIVAVLIA), plexin A2 (SEQ ID NO. 11:LLTLPAIVSIAAGGSLLLIIVIIVLIAY), plexin A3 (SEQ ID NO. 12:LTLPAMMGLAAGGGLLLLAITAVLVA), plexin A4 (SEQ ID NO. 6:LSLPAIVSIAVAGGLLIIFIVAVLIA), Nr-CAM (SEQ ID NO. 7:GWFIGLMCAVALLILILLIVCFI), L1-CAM (SEQ ID NO. 13: GWFIGFVSAIILLLLVLLIL),integrin beta1 (SEQ ID NO. 9: IIPIVAGVVAGIVLIGLALLLIW) or integrin beta2(SEQ ID NO. 14: IAAIVGGTVAGIVLIGILLLVIW) transmembrane domain relates toamino acid sequence having an identity of more than 60% with saidtransmembrane domains or fragments thereof, for example of more than 70%or of more than 80%, preferably of more than 85%, most preferably ofmore than 90% and advantageously of more than 95%.

As used herein, “a fragment of a transmembrane domain” relates to apolypeptide which is more than 10 amino acids length, preferably morethan 14 amino acids length, as an example more than 18 amino acidslength, and more preferably more than 22 amino acids.

The identity differences between the above described transmembranedomains and the amino acid sequence of the peptidic antagonist of theinvention result from amino acids substitution in the transmembranedomain amino acid sequences of the peptidic antagonist.

Preferably, the substituted amino acid(s) in these transmembrane domainsis (are) neutral and/or hydrophobic amino acids, and most preferablyhydrophobic amino acids. Such neutral and hydrophobic amino acids arewell known from one of skill in the art.

According to a specific embodiment, said transmembrane domain amino acidsequences have an identity of 100% with said transmembrane domains orfragments thereof.

In a second aspect the present invention relates to a nucleic acidencoding for a peptidic antagonist as described above.

Said nucleic acid corresponds to RNA or DNA, preferably to DNA.

According to a preferred embodiment, the nucleic acid encoding thepeptidic antagonist is operatively linked to a gene expression sequence,which directs the expression of nucleic acid within a prokaryotic or aneukaryotic cell, preferably an eukaryotic cell. The “gene expressionsequence” is any regulatory nucleotide sequence, such as a promotersequence or promoter-enhancer combination, which facilitates theefficient transcription and translation of the peptidic antagonistnucleic acid to which it is operatively linked and any signal sequenceensuring appropriate targeting of the peptidic antagonist to the plasmamembrane. The gene expression sequence may, for example, be a mammalianor viral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPTR), adenosine deaminase, pyruvate kinase, beta.-actinpromoter, muscle creatine kinase promoter, human elongation factorpromoter and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the simian virus (e.g., SV40), papilloma virus,adenovirus, human immunodeficiency virus (HIV), cytomegalovirus (CMV),Rous sarcoma virus (RSV), hepatitis B virus (HBV), the long terminalrepeats (LTR) of Moloney leukemia virus and other retroviruses, and thethymidine kinase promoter of herpes simplex virus. Others constitutivepromoters are known to those of ordinary skill in the art. The promotersuseful as gene expression sequences of the invention also includeinducible promoters. Inducible promoters are expressed in the presenceof an inducing agent. For example, the metallothionein promoter isinduced to promote transcription and translation in the presence ofcertain metal ions. Others inducible promoters are known to those ofordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined antigen nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

As used herein, the peptidic antagonist nucleic acid sequence and thegene expression sequence are said to be “operably linked” when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the peptidic antagonist codingsequence under the influence or control of the gene expression sequence.Two DNA sequences are said to be operably linked if induction of apromoter in the 5′ gene expression sequence results in the transcriptionof the peptidic antagonist sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the peptidic antagonist sequence,or (3) interfere with the ability of the corresponding RNA transcript tobe translated into a protein. Thus, a gene expression sequence would beoperably linked to a peptidic antagonist nucleic acid sequence if thegene expression sequence were capable of effecting transcription of thatantigen nucleic acid sequence such that the resulting transcript istranslated into the desired protein or polypeptide.

The peptidic antagonist nucleic acid may be delivered in vivo alone orin association with a vector. In its broadest sense, a “vector” is anyvehicle capable of facilitating the transfer of the peptidic antagonistnucleic acid to the cells and preferably cells expressing neuropilins.Preferably, the vector transports the nucleic acid to cells with reduceddegradation relative to the extent of degradation that would result inthe absence of the vector. The vector optionally includes theabove-described gene expression sequence to enhance expression of thepeptidic antagonist nucleic acid in neuropilins' expressing cells. Ingeneral, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the peptidic antagonist nucleic acid sequences.Viral vectors are a preferred type of vector and include, but are notlimited to nucleic acid sequences from the following viruses:retrovirus, such as moloney murine leukemia virus, harvey murine sarcomavirus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus,adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barrviruses; papilloma viruses; herpes virus; vaccinia virus; polio virus;and RNA virus such as a retrovirus. One can readily employ other vectorsnot named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in KRIEGLER (ALaboratory Manual,” W.H. Freeman C.O., New York, 1990) and in MURRY(“Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Cliffton,N.J., 1991).

Preferred viruses for certain applications are the adeno-viruses andadeno-associated viruses, which are double-stranded DNA viruses thathave already been approved for human use in gene therapy. Theadeno-associated virus can be engineered to be replication deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g., SANBROOK et al., “Molecular Cloning: A LaboratoryManual,” Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been used as DNA vaccines fordelivering antigen-encoding genes to cells in vivo. They areparticularly advantageous for this because they do not have the samesafety concerns as with many of the viral vectors. These plasmids,however, having a promoter compatible with the host cell, can express apeptide from a gene operatively encoded within the plasmid. Somecommonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, andpBlueScript. Other plasmids are well known to those of ordinary skill inthe art. Additionally, plasmids may be custom designed using restrictionenzymes and ligation reactions to remove and add specific fragments ofDNA. Plasmids may be delivered by a variety of parenteral, mucosal andtopical routes. For example, the DNA plasmid can be injected byintramuscular, intradermal, subcutaneous, or other routes. It may alsobe administered by intranasal sprays or drops, rectal suppository andorally. It may also be administered into the epidermis or a mucosalsurface using a gene-gun. The plasmids may be given in an aqueoussolution, dried onto gold particles or in association with another DNAdelivery system including but not limited to liposomes, dendrimers,cochleate and microencapsulation.

The nucleic acid vector can include selectable markers that are activeboth in bacteria and in mammalian cells.

According to a first specific embodiment, the nucleic acid vector of thepresent invention corresponds to “naked DNA” like plasmids, cosmids orphagemids. Such naked DNA can be associated with non-lipid cationicpolymers (WU and WU, J. Biol. Chem., vol. 263, p: 14621-4, 1988) orliposomes (BRIGHMAN et al., Am. J. Med. Sci., vol. 298, p: 278-81, 1989)to form complexes enhancing cellular uptake.

According to a second specific embodiment, the nucleic acid vector is aviral vector adapted for in vivo gene therapy protocols. Examples ofappropriate viral vectors includes retroviral vectors as described in EP0871459, EP 0386882 and EP 1222300 and adenovirus vectors as describedin US 2004/265273 and U.S. Pat. No. 6,638,502. In this case, theinternalization of virus occurs through the specific interaction of theviral envelope with a cell surface receptor, followed byreceptor-mediated endocytosis of the virus/receptor complex.

In a third aspect the present invention relates to a compositioncomprising a peptidic antagonist as described above, a nucleic acidencoding thereof, or a nucleic acid vector comprising said nucleic acid,eventually associated with a pharmaceutically acceptable vehicle.

As an example of pharmaceutically acceptable vehicle, the compositionmay comprise emulsions, microemulsions, oil-in-water emulsions,anhydrous lipids and oil-in-water emulsions, other types of emulsions.The composition may also comprise one or more additives (e.g., diluents,excipients, stabilizers, preservatives). See, generally, Ullmann'sEncyclopedia of Industrial Chemistry, 6^(th) (various editors,1989-1998, Marcel Dekker); and Pharmaceutical Dosage Forms and DrugDelivery Systems (ANSEL et al., 1994, WILLIAMS & WILKINS).

Advantageously, said composition comprise a concentration of saidpeptidic antagonist of more than 10⁻¹² M, preferably more than 10⁻¹¹ Mand most preferably more than 10⁻¹⁰ M.

Peptidic antagonists, nucleic acids or nucleic acid vectors may besolubilized in a buffer or water or incorporated in emulsions andmicroemulsions. Suitable buffers include, but are not limited to,phosphate buffered saline Ca⁺⁺/Mg⁺⁺ free (PBS), phosphate bufferedsaline (PBS), normal saline (150 mM NaCl in water), Tris buffer andsurfactants.

There are numerous causes of peptide instability or degradation,including hydrolysis and denaturation. Hydrophobic interaction may causeclumping of molecules together (i.e. aggregation). This result mayentail diminution of the induction of a Treg response. Stabilizers maybe added to lessen or prevent such problems.

Stabilizers include cyclodextrine and derivatives thereof (see U.S. Pat.No. 5,730,969). Suitable preservatives such as sucrose, mannitol,sorbitol, trehalose, dextran and glycerin can also be added to stabilizethe final formulation. A stabilizer selected from ionic and non-ionicsurfactants, D-glucose, D-galactose, D-xylose, D-galacturonic acid,trehalose, dextrans, hydroxyethyl starches, and mixtures thereof may beadded to the formulation. Addition of alkali metal salt or magnesiumchloride may stabilize a peptide. The peptide may also be stabilized bycontacting it with a saccharide selected from the group consisting ofdextran, chondroitin sulphuric acid, starch, glycogen, dextrin, andalginic acid salt. Other sugars that can be added includemonosaccharides, disaccharides, sugar alcohols, and mixtures thereof(E.g., glucose, mannose, galactose, fructose, sucrose, maltose, lactose,mannitol, xylitol). Polyols may stabilize a peptide, and arewater-miscible or water-soluble. Suitable polyols may be polyhydroxyalcohols, monosaccharides and disaccharides including mannitol, glycrol,ethylene glycol, propylene glycol, trimethyl glycol, vinyl pyrrolidone,glucose, fructose, arabinose, mannose, maltose, sucrose, and polymersthereof. Various excipients may also stabilize peptides, including serumalbumin, amino acids, heparin, fatty acids and phospholipids,surfactants, metals, polyols, reducing agents, metal chelating agents,polyvinyl pyrrolidone, hydrolysed gelatin, and ammonium sulfate.

In a fourth aspect the present invention relates to a method ofprophylactic or therapeutic treatment of a subject suffering from adisease associated with class III semaphorins/neuropilins complexessignal transduction pathways comprising the step of administrating acomposition as described above to said subject.

As used herein, the term “subject” denotes a Mammal, such as a rodent, afeline, a canine and a primate. The subject is an animal such as cow,pig, horse, cat, dog and most preferably a human.

A disease associated with class III semaphorins/neuropilins complexessignal transduction pathways can be simply determined by one of skill inthe art. As an example of such diseases, one can cite neurodegenerativediseases (like Alzheimer disease, Parkinson disease, central nervoussystem lesions, demyelination associated pathologies), cancers (likelung, breast and mesothelial cancers, carcinoma or glioma), and alldiseases associated to abnormal angiogenesis.

Advantageously, said administration of said composition corresponds to aconcentration of said peptidic antagonist of more than 10⁻¹² M,preferably more than 10⁻¹¹ M and most preferably more than 10⁻¹⁰ M.

In a fifth aspect the present invention relates to the use of a peptidicantagonist as described above, a nucleic acid encoding thereof, or anucleic acid vector comprising said nucleic acid for the manufacture ofa medicament for the prevention or treatment of a subject suffering of adisease associated with class III semaphorins/neuropilins complexessignal transduction pathways.

A disease associated with class III semaphorins/neuropilins complexessignal transduction pathways can be simply determined by one of skill inthe art. As an example of such diseases, one can cite neurodegenerativediseases (like Alzheimer disease, Parkinson disease, central nervoussystem lesions, demyelination associated pathologies), cancers (likelung, breast and mesothelial cancers, carcinoma or glioma), and alldiseases associated to abnormal angiogenesis.

In a preferred embodiment, the present invention relates to the use of apeptidic antagonist as described above, a nucleic acid encoding thereof,or a nucleic acid vector comprising said nucleic acid for themanufacture of a medicament for the prevention or treatment of a subjectsuffering of a neurodegenerative disease selected in the groupcomprising Alzheimer disease, Parkinson disease, central nervous systemlesions and demyelination associated pathologies.

In a second preferred embodiment, the present invention relates to theuse of a peptidic antagonist as described above, a nucleic acid encodingthereof, or a nucleic acid vector comprising said nucleic acid for themanufacture of a medicament for the prevention or treatment of a subjectsuffering of a cancer selected in the group comprising lung cancer,breast cancer, mesothelial cancers, carcinoma and glioma

In a third preferred embodiment, the present invention relates to theuse of a peptidic antagonist as described above, a nucleic acid encodingthereof, or a nucleic acid vector comprising said nucleic acid for themanufacture of a medicament for the prevention or treatment of a subjectsuffering of a disease associated with abnormal angiogenesis.

Advantageously, said medicament allows the release of a concentration ofsaid peptidic antagonist of more than 10⁻¹² M, preferably more than10⁻¹¹ M and most preferably more than 10⁻¹⁰ M.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequences of the transmembrane domains ofmouse neuropilin-1, neuropilin-1, plexin A1, plexin A2, plexin A3,plexin A4, Nr-CAM, L1-CAM, integrin beta 1, and integrin beta 2.

FIG. 2 shows the amino acid sequences of the transmembrane domains ofhuman neuropilin-1, neuropilin-1, plexin A1, plexin A2, plexin A3,plexin A4, Nr-CAM, L1-CAM, integrin beta 1, and integrin beta 2.

FIG. 3(A) is a graph showing the dimerization capacity of thetransmembrane domain of neuropilin-1 compared to that of thetransmembrane domain of EGF receptor, Erb-2 protein and glycophorin Ameasured as described in Example 1. FIG. 3(B) is a model showing theexistence of a spatial compact organization of the transmembrane domainof neuropilin-1 presenting inter-helices interactions in favor of dimerformation.

FIG. 4(A) is a graph showing the effect of increasing the concentrationof the transmembrane domain of neuropilin-1 (pTM-NRP1) on cortical axonscollapses triggered by Sema3A. FIG. 4(B) is a graph showing the effectof pTM-NRP1 (10⁻⁸M) or of the transmembrane domain of neuropilincontaining a triple G->V mutation (pTM-NRP1^(mut)) (10⁻⁸M) on corticalaxons collapses triggered by Sema3A.

FIG. 5(A) shows the morphology of COSs-1 cells expressing neuropilin-1and plexin-A1 in the presence or absence of Sema3A with or withoutpTM-NRP1 or pTM-NRP1^(mut). FIG. 5(B) is a graph showing the effects ofpTM-NRP1 or pTM-NRP1^(mut) on cellular collapses triggered by Sema 3A.

FIG. 6(A) shows wild type COS cells (control) and NRP1-expressing COScells (COS-NRP1) after incubation with AP-Sema3A with or withoutpTM-NRP1 or pTM-NRP1^(mut). FIG. 6(B) shows the mean intensity ofoptical density per cell measured for the samples described in FIG.6(A).

FIG. 7(A) shows wild type COS cells (control), NRP1-expressing COS cells(COS-NRP1), and COS cells expressing neuropilin with a triple G->Vmutation (COS-NRP1^(mut)) after incubation with AP-SemaA3 with orwithout pTM-NRP1. FIG. 7(B) shows the mean intensity of optical densityper cell measured for the samples described in FIG. 7(A).

FIG. 8(A) shows results of a differentiation assay on PC12 cells afterincubation with or without NGF (100 ng/mL), Sema 3A, or pTM-NRP1 (10⁻⁹M). FIG. 8(B) shows the percentage of differentiation for each of thesamples described in FIG. 8(A).

FIG. 9 is a graph showing the binding of AP-Sema3A to its receptor inthe presence of pTM-NRP1 peptide (black bars) or mutated pTM-NRP1 (greybars) (*: p<0.005; **: p<0.01, Student test).

FIG. 10 is a graph showing the formation of NRP1 complexes in C6 cellsexpressing NRP1 and plexin-A1 in the presence or in the absence of thetransmembrane domain of NRP1 (pTM-NRP1) and of the ligand Sema3A.

FIG. 11 shows several mouse brain sections after injection into thebrain of C6 cells with or without pTM-NRP1 or pTM-NRP1^(mut) peptides.The positions of the tumor, cortex, striatum, corpus callosum (cc),hippocampus (Hp) and lateral ventricle (VL) are depicted.

FIG. 12 shows mouse brain sections after injection into the brain of C6cells with or without pTM-NRP1 or pTM-NPR1^(mut) peptides afterimmunostaining of CD34.

FIG. 13 shows tumor cell aggregates in the presence or not of VEGF₁₆₅(50 ng/ml), pTM-NRP1 or pTM-NRP1^(mut) (10⁻⁸ M).

The invention is further illustrated below by the following Examples,which are not intended to limit its scope.

EXAMPLES 1) The Transmembrane Domain of the NRP1 Receptor has aDimerization Capacity

The ToxLuc System derived from the ToxCat system described in RUSS andENGELMAN (Proc. Natl. Acad. Sci. USA, vol. 96, p: 863-8, 1999) has beenused to investigate NRP1 transmembrane domain-mediated dimerization.This system enable to measure transmembrane helix-helix oligomerizationin E. Coli internal membrane. The dimerization capacity of thetransmembrane domain of NRP1 (SEQ ID NO. 1, ILITIIAMSALGVLLGAVCGVVL) hasbeen compared with the one of EGF receptor (SEQ ID NO. 17,SIATGMVGALLLLLVVALGIGLFM), Erb-2 protein (SEQ ID NO. 18,SIISAVVGILLVVVLGVVFGILI) and glycophorin A (SEQ ID NO. 19,ITLIIFGVMAGVIGTILLISYGI).

Several constructions were performed, which encodes for the specificfusion proteins. These fusion proteins comprised the N-terminal DNAbinding domain of ToxR (a dimerization-dependent transcriptionalactivator) fused to the transmembrane domain of NRP1, EGF receptor, Erb2receptor and glycophorin A respectively, and a monomeric periplasmicanchor (the Maltose Binding Protein: MBP).

The TM sequences of interest were expressed in the bacteria DH5a (MM39)as chimeric proteins flanked by ToxR and by the maltose binding protein(MBP). TM domain-mediated oligomerization results in ToxR-activatedexpression of a reporter gene encoding chloramphenicol acetyltransferase(CAT) in the original version of the system. For convenience, we usedconventional molecular biology methods to replace the initial CAT geneby that of luciferase. Synthetic TM sequences corresponding toneuropilin, EGF receptor and wild-type erbB2 were cloned into the newplasmid as NheI/DpnII fragments. Chimeras with TM sequences derived fromglycophorin A or its G83I mutant (RUSS and ENGELMAN, Proc. Natl. Acad.Sci. USA., vol. 96(3), p: 863-8, 1999) were used as controls. Luciferaseassay was performed using the ROCHE assay kit according to themanufacturer instructions, and a Berthold Microlumat plate luminometer.

The results are shown in FIG. 3A for the different constructions.

The results show that the bacteria transformed with the constructcontaining the transmembrane domain of NRP1 show a significantly higherluciferase luminescence than those containing the transmembrane domainof Erb-2 (4.7 factor) or EGFR (6.1 factor) and slightly superior tothose containing the one of glycophorin-A (1.2 factor). Interestingly,we confirmed the dimerization capacity of NRP1 TM domain by a 3dimensional model of TM-NRP1 obtained with the SwissPdbViewer softwareon the basis of the RMN structure of the glycophorin-A. This theoreticalapproach, although being minimalist, confirmed the existence of aspatial compact organization of the TM-NRP1 presenting inter-helicesinteractions in favor of dimer formation (FIG. 3B).

In conclusion, the transmembrane domain of NRP1 is able to induce thedimerization with a efficiency stronger than the dimerization capacityof GpA transmembrane domain.

2) The Transmembrane Domain Peptide of the NRP1 Receptor InhibitsCortical Axons Collapses Triggered by Sema3A

The inhibitory axon growth property of Sema 3A is supposed to be linkedto its induction capacity of growth cone collapses. To investigate theeffect of the pTM-NRP1 peptide on the Sema 3A functional properties, thegrowth of cortical neurons was analyzed in the presence or not of Sema3A and of pTM-NRP1 or pTM-NRP1^(mut) (NRP1 TM with a triple G->Vmutation) peptides.

Laminine/Poly-L-Lysine substrates have been made by adding 980 μL ofGey's balanced salt solution (GBSS, SIGMA) to 10 μL laminine (lmg/ml,SIGMA) and to 10 μL poly-L-Lysine (10 mg/ml, SIGMA). Sterile coverslipshave been placed in a big dish and added with substrate (100 μL). Then,‘sandwiches’ have been prepared by covering each coverslip with a secondone. After an incubation for at least 30 min at 37° C. under 5% CO₂ inair, ‘sandwiches’ have been opened and each coverslip has been rinsedwith deionized water. Explants have been cultured on dried coverslips.

Neocortex fragments prepared from E15 mouse embryos (E1 determined asthe first day of embryonic development by detection of vaginal plug)have been transferred on a tissue chopper disk. Tissue have been cutinto 200×200 μm by rotating the disk by 90° after the first cut. Cortexcubes have been collected with a spatula and put into culture medium ina Petri dish. Two coverslips with substrate have been placed in a smallPetri dish (50 mm diameter, FALCON). After adding 750 μl of culturemedium, the coverslips should stay in the incubator for at least 10minutes. Using a dissecting microscope, forty to fifty cortical explantshave been collected in 20 μl culture medium and carefully placed ontothe coverslips. After 15 min at room temperature, most explants haveadhered to their substrate. 2250 μl of culture medium have been slowlyadded to each dish. Then, explants cultures have been kept at 37° C.under 5% CO₂ in air.

A radial outgrowth could be seen after 18-24 h in culture, andindividual fibres and growth cones could have therefore been analyzed.Products tested on growth cones have been directly added in culturemedium for 2 h:

-   -   purified Sema3A (100 ng/ml) prepared from conditioned medium of        HEK293 cells stably expressing Sema3A. The purification was        performed by using the anti-Flag system, SIGMA)    -   pTM-NRP1 (10⁻⁸, 10⁻⁹, 10⁻¹⁰ and 10⁻¹¹ M),    -   10⁻⁸M mpTM-NRP1,    -   10⁻⁸M pTM-ErbB2w (SEQ ID NO. 18).

After incubation, 4% formaldehyde has been directly added in culturemedium (v/v) for 15 min. Then the solution has been removed and replacedby 4% formaldehyde for 15 min.

The FIG. 4A shows the effect of increasing concentrations of pTM-NRP1peptide (10⁻⁸, 10⁻⁹, 10⁻¹⁰ and 10⁻¹¹M) on cortical axons collapsestriggered by Sema3A.

The FIG. 4B shows the effect of pTM-NRP1 or pTM-NRP1^(mut) peptide (10⁻⁹M) on cortical axons collapses triggered by Sema3A.

The results show that more than 50% of collapsed cortical axons wereobserved in Sema3A-treated cells, whereas less than 10% of the corticalaxons presented collapsed morphology in control conditions (FIG. 4A).Furthermore, the addition of increasing concentrations of the wild typepTM-NRP1 peptide suppressed the collapsed effect of Sema3A on corticalaxons in a dose dependant manner with an IC50 of nearly 10⁻¹¹ M.

In contrast, the addition of the pTM-NRP1^(mut) peptide did not blockthe cortical axons collapses triggered by Sema3A (FIG. 4B).

Control experiment with peptide dilution buffer and with ErbB2 peptide,which contains a GxxxG motif, show no effect on the collapsing effect ofSema 3A.

These results demonstrate that the addition of a synthetic peptidemimicking the transmembrane domain of NRP1 abolishes the effects ofSema3A on cortical axons, inhibiting cortical axons collapses. Moreover,this biological activity is associated with the GxxxG motif and isspecific from the pTM-NRP1 peptide.

3) The Transmembrane Domain Peptide of the NRP1 Receptor Inhibits COSCellular Collapses Triggered by Sema3A

The COS cells do not express semaphorin receptors and are thereforenaturally not sensitive to these guidance signals. Nevertheless, theartificial expression of NRP1 and Plexin-A1 in COS cells allows Sema 3Ato trigger cellular collapses. To investigate the effect of the pTM-NRP1peptide on the Sema3A functional properties, the shape of COS cellsexpressing NRP1 and Plexin-A1 was analyzed in the presence or not ofSema 3A and of pTM-NRP1 or pTM-NRP1^(mut) peptide.

COS-1 cells have been transfected by 1 μg of pBK-CMV (STRATAGENE)plasmids containing NRP1 and plexin-A1 coding sequences (provided by Pr.PUSCHEL; MUNSTER Universitat, Germany) with LIPOFECTAMINE 2000 (INVITROGEN) according to the manufacturer's instructions in 6-well plates.Stably COS-1 transfected cells have been selected with 0.7% geneticine.Stably transfected COS-1 cells have been cultured on 12-well plates withpreviously poly-L-lysine-coated glass coverslips. Cells have been thenincubated 1 hour with pTM-NRP1 or pTM-NRP1^(mut) peptide (10⁻⁹M) at 37°C. The culture medium has been then removed and replaced by conditionedmedium of HEK cells stably transfected or not with a constructionexpressing Sema 3A (100 μl/ml D-MEM) for 4 hours at 37° C. Finally,cells have been fixed with 2% formaldehyde for 30 minutes followed by 15minutes in formaldehyde 4%. For each condition tested, about 400 cellshave been analyzed.

The FIG. 5A show the morphology of COS-1 cells expressing NRP1 andPlexin-A1 in the presence or absence of Sema 3A with or without pTM-NRP1or pTM-NRP1^(mut) peptide. The FIG. 5B show the effect of pTM-NRP1 orpTM-NRP1^(mut) peptide on the cellular collapses triggered by Sema 3A(*: p<0.001).

The results show that more than 50% of collapsed cells were observed inSema 3A-treated cells, whereas less than 10% of the cells presentedcollapsed morphology in control conditions (FIG. 5B). Furthermore, theaddition of 10⁻⁹ M of pTM-NRP1 peptide completely abolished thecollapsing effect of Sema3A on COS-1 cells. In contrast, the addition of10⁻⁹ M of the pTM-NRP1^(mut) peptide, which has a mutated GxxxG motif,did not block the cellular collapses triggered by Sema 3A.

These results demonstrate that the addition of a synthetic peptidemimicking the transmembrane domain of NRP1 abolishes the cellularcollapsing effect of Sema 3A with its GxxxG motif.

To address the mechanism by which pTM-NRP1 blocked Sema3A signaling weperformed binding assays.

NRP1 expressing COS cells were incubated with AP-Sema3A, a fusionprotein of Sema3A and the secreted alkaline phosphatase (BAGNARD et al.,1998).

Wild-type COS cells or NRP1-expressing COS cells (COS-NRP1) werecultured on 12-wells plates on poly-L-lysine-coated glass coverslips(0.005 mg/ml). After one-hour incubation with pTM-NRP1 or pTM-NRP1^(mut)(10⁻⁹M) in serum-free medium at 37° C., the culture medium was replacedby conditioned medium containing alkaline phosphatase-coupled Sema3A(AP-Sema3A; BAGNARD et al., 1998) obtained from AP-Sema3A stablyexpressing HEK cells for 90 mn. Conditioned medium without semaphorinserved as a control (obtained from non-transfected HEK). Cells werewashed three times with PBS and fixed in 4% formaldehyde before transferin a new dish. After three washes in PBS, the plate was warmed for 50 mnat 65° C. Cells were subsequently incubated with 1 ml of alkalinephosphatase substrate (NBT/BCIP, SIGMA) in the dark. After 45 mn,substrate was removed and glass coverslips were rinsed. Pictures wereacquired with a conventional microscope and analyzed with AxioVision LEZeiss software. For each condition tested, about 60 cells were analyzedto determine binding levels as a function of optical density.Statistical analysis was performed by using a Student's t test.

The FIG. 6A shows the Wild type COS cells (control) or NRP1 expressingCOS cells (COS-NRP1) after incubation with AP-Sema3A with or withoutpTM-NRP1 or pTM-NRP1^(mut).

The FIG. 6B shows the mean intensity of optical density per cell for theprevious tested conditions.

The results show that the binding of AP-Sema3A was significantly reducedby addition of pTM-NRP1 (FIGS. 6A and B). Strikingly, the addition ofpTM-NRP1^(mut) did not block AP-Sema3A binding to COS cells.

These results demonstrate that the GxxxGxxxG domain of NRP1 TM appearedcrucial to trigger Sema3A binding and subsequent inhibitory effect.

4) Mutation of the TM Domain of NRP1 Disrupts Receptor Function

In order to confirm the pivotal role of the GxxxGxxxG motif, mutationswere introduced into the TM domain of a full length NRP1 to replace allthree glycines residues by valines (NRP1^(mut)) as in the mutatedpeptide.

A plasmid encoding for a NRP1 protein with the triple (G->V) mutation inthe transmembrane region was transfected in COS cells as describedpreviously. Then, binding experiments were conducted in COS cellsexpressing this mutated form of NRP1 as previously.

The FIG. 7A shows the Wild type COS cells (control), NRP1 expressing COScells (COS-NRP1), or NRP1 with the triple (G->V) mutation expressing COScells (COS-NRP1^(mut)) after incubation with AP-Sema3A with or withoutpTM-NRP1.

The FIG. 7B shows the mean intensity of optical density per cell for theprevious tested conditions.

The results show that, while significant binding was observed in COScells expressing the wild type NRP1 very low if any binding of AP-Sema3Awas detected in cells expressing NRP1^(mut) (FIG. 7A). The strongreduction of binding was similar to the one obtained in the presence ofthe pTM-NRP1. Strikingly, when this NRP1 mutant (NRP1^(mut)) wasexpressed in COS cells together with PlexA1, Sema3A was no longer ableto induce a cell collapse (FIG. 7B). This further confirmed theimportance of the GxxxGxxxG motif of NRP1 TM domain for the formation ofa functional Sema3A receptor.

5) Synthetic Peptides Mimicking TM-NRP1 Alter the Formation of theSemaphorin Receptor Complex

To further investigate the biochemical consequence of the TM-NRP1 interms of receptor complex formation, we analyzed the formation ofcomplexes in the PC12 neuronal cells model. Interestingly, Sema3A hasbeen shown to promote the growth of neurites in these cells through aNGF-independent pathway (SCHWAMBORN et al., J. Biol. Chem., vol.279(30), p: 30923-6, 2004).

PC12 (ATCC: CRL-1721) were grown in D-MEM medium with 4.5 g of glucose/L(GIBCO), 5% FVS, 10% horse serum, glutamine 580 mg/L and antibiotics.For functional assays, PC12 were cultured on 12-wells plates withpreviously poly-L-lysine-coated glass coverslips. PC12 cells wereincubated 1 h with or without pTM-NRP1 peptide (10⁻⁹M) at 37° C. Culturemedium was removed and replaced by NGF-containing (100 ng/mL; GIBCO)serum free medium or by conditioned medium obtained from HEK293 cellsstably expressing Sema3A or non-transfected cells (control, see BAGNARDet al., 1998 for details) for 12 h at 37° C.

Cells were fixed with 2% formaldehyde for 30 mn followed by 15 mn in 4%formaldehyde. For each condition tested, around 400 cells were analyzedto evaluate neuritic outgrowth (Statistical analyses were made by usingχ² test).

The FIG. 8A shows the result of the differentiation assay of PC12 cellsafter their incubation with or without NGF (100 ng/ml), Sema 3A,pTM-NRP1 (10⁻⁹ M).

The FIG. 8B shows the percentage of differentiated cells for eachcondition.

The results show that the addition of NGF or Sema3A induced PC12 celldifferentiation without synergistic effects (FIGS. 8A and 8B). Moreover,when experiments were done in the presence of pTM-NRP1, the Sema3Ainduced neurite growth promotion was significantly reduced while the NGFeffect was preserved. This demonstrated that the addition of the peptidespecifically blocked the activation of Sema3A-dependent pathways withoutaffecting other signaling pathways.

6) The Transmembrane Domain Peptide of the NRP1 Receptor Antagonizes theBinding of the Ligand Sema3A to its Receptor NRP1

In order to investigate the NRP1 transmembrane domain role in thelinkage NRP1-class III semaphorins, the binding of the ligand Sema3A toits receptor NRP1 on glioma cells was measured in the presence or not ofthe transmembrane domain peptide of NRP1 (pTM-NRP1 peptide; SEQ IDNO. 1) or of a mutated peptide (mpTM-NRP1; SEQ ID NO. 20,ILITIIAMSALVVLLVAVCVVVLYRKR). These pTM-NRP1 Peptides have beensynthesized by automatic peptidic synthesis (Fmoc chemistry, APPLIEDSYSTEM), and analyzed by mass spectrometry. Peptides purity has beenestimated by RP-HPLC (BECKMAN) as higher than 90%.

Rat C6 glioma cells, which express semaphorin receptors, has been usedto determine the binding capacity of AP-Sema3A, a secreted alkalinephosphatase version of Sema 3A (ADAMS et al., 1997, BAGNARD et al.,1998). These cells were grown and plated in MEM medium (GIBCO) with 10%foetal calf serum (PERBIO), glutamine 0.5 mM (GIBCO) and antibiotics:100 U/ml penicillin and 100 ng/ml streptomycin (SIGMA).

C6 Cells were cultured in 96-well plates and incubated with or withoutfreshly diluted pTM-NRP1 or pTM-NRP1^(mut) peptides (10⁻¹² M to 10⁻¹⁰ M)for 1 hour at 37° C. The culture medium has been then replaced byconditioned medium of HEK cells stably transfected with a constructionexpressing AP-Sema 3A (ADAMS et al., EMBO J., vol. 16(20), p: 6077-86,1997; BAGNARD et al., 1998). Conditioned medium from HEK non transfectedcells has been used as an internal control. The pTM-NRP1 Peptide hasbeen synthesized by automatic peptidic synthesis (Fmoc chemistry,APPLIED SYSTEM), and analyzed by mass spectrometry. Peptides purity hasbeen estimated by RP-HPLC (BECKMAN) as higher than 90%., the cells havebeen washed with PBS and incubated with 50 μl of alkaline phosphataseluminescent substrate (AMERSHAM). The luminescence has been read after15 minutes with MICROLUMAT PLUS system (BERTHOLD TECHNOLOGIES) accordingto the manufacturer's instructions. Experiments were done 4 times foreach peptide.

The results for the AP-Sema 3A binding to its receptor in the presenceof pTM-NRP1 peptide (black bar) or mutated (grey bar) are shown in FIG.9 (*: p<0.005; **: p<0.01, Student t test).

The results show that the binding of AP-Sema3A to its receptor NRP1 onC6 cells was blocked in a dose dependent manner by addition ofincreasing concentrations of the wild type pTM-NRP1 peptide. In presenceof 10⁻¹⁰M of the wild type pTM-NRP1 peptide, the binding of AP-Sema3A toNRP1 decreased from about 50% compared to the absence of peptide. Incontrast to the wild type pTM-NRP1 peptide, addition of pTM-NRP1^(mut)peptide did not block the binding of AP-Sema3A to NRP1.

As a result, the binding of Sema3A to its receptor NRP1 involves thetransmembrane domain of NRP1 and requires the integrity of the doubleGxxxG motif.

7) The Transmembrane Domain Peptide of the NRP1 Receptor Alters theFormation of the Semaphorin Receptor Complex

To further investigate the role of the transmembrane domain of the NRP1receptor in the formation of the NRP1 receptor complex, the formation ofNRP1 complexes has been determined on C6 cells expressing NRP1 andplexin-A1 in the presence or not of the transmembrane domain peptide ofNRP1 (pTM-NRP1 peptide) and of the ligand Sema3A.

C6 cells expressing NRP1 and Plexin-A1 were incubated or not with thepTM-NRP1 peptide (10⁻⁹ M) for 1 h. The culture medium has been thenreplaced of HEK cells stably transfected or not with a constructionexpressing Sema 3A. Confluent C6 have been harvested with 10 mM EDTA andcentrifuged. The pellet has been washed in PBS and then diluted in lysisbuffer (Tris-HCL/NaCl; 50/150; pH8.0) with 0.1% SDS, 1 mM EDTA, 1%NP-40, 0.5% DOC, 2 mM vanadate and proteases inhibitors without SDS(PIERCE). After solubilization for 1 h at 4° C., protein amount isestimated by bicinchoninic acid method (BCA Protein Assay, PIERCE).

Sucrose density gradient sedimentation experiments were based on a stepgradient containing 25%, 17%, 10% and 3% sucrose. Solutions have beenmade from Hepes/NaCl buffer (30/30, pH 7.6, 0.12% triton) and Hepes/NaClbuffer with 1M sucrose. These solutions were successively loaded inorder to form a linear gradient (LERAY et al., Arch Biochem Biophys.,1992).

The cell lysates were placed on the gradient in an ultracentrifuge tubeand centrifuged at 100 000 g for 1 hour with a TL-100 ultracentrifuge(BECKMAN) and the fractions have been collected from the bottom (13drops/fraction).

According to LAEMMLI's method, an equivalent volume of loading bufferhas been added to samples (62.5 mM Tris-HCL pH 6.8, 10% glycerol, 2%SDS, DTT, bromophenol blue) and these have been boiled for 10 minutes.Then samples have been subjected to SDS-PAGE on acrylamide gel (5-20%)at constant voltage and temperature in adequate buffer (0.025M Tris,0.192M Glycine pH 8.3, 0.01% SDS). Proteins have been then transferredto methanol-activated polyvinyldiene difluoride (PVDF) membrane at 4° C.for 3 hours in a buffer containing 20% ethanol, 0.025M Tris, 0.192MGlycine pH 8.3, and 0.01% SDS. Finally, the PVDF membrane has beenblocked for 1 hour with PBS/BSA 5%.

The membrane has been then incubated 2 hours with polyclonal anti-NRP1at a 1:1000 dilution (ONCOGENE). The membrane has been washed threetimes in PBS/0.2% TWEEN 20 and incubated with the secondary antibody(A/G protein, PIERCE, 1:100 000 or horseradish peroxydase-linkedanti-rabbit IgG, AMERSHAM, 1:500). Immunoreactivity has been thendetected with an enhanced chemoluminescence western blot detectionsystem (PIERCE) according to the manufacturer's instructions.

The results are shown in FIG. 10. NRP1 percentage in each fraction wascalculated from total revealed NRP1. In this figure, heavy fractions ofthe sucrose gradient containing oligomers including Plexin-A1 correspondto black bars, medium fractions containing NRP1 dimers correspond togrey bars, and light fractions were almost composed of NRP1 monomerscorrespond to empty bars.

The results show that in the absence of the ligand Sema 3A, NRP1 waspredominantly detected in the medium fractions of the gradient sucrose.Thus, the NRP1 dimers represented the major forms of NRP1 receptor inthe absence of its ligand Sema3A.

In contrast, in presence of Sema3A, NRP1 receptors were predominantlypresent as oligomeric forms including plexin-A1 in the heavy fractions.

The addition of the pTM-NRP1 peptide in the presence of Sema 3A modifiedthe distribution of NRP1, which was mainly detected in the lightfractions corresponding to the migration level of NRP1 monomers. Thus,the oligomerization of NRP1 was inhibited by the presence of thepTM-NRP1 peptide. Hence, the transmembrane domain of NRP1 is involved inthe formation of NRP1 receptor complex. Consequently, the decrease ofSema 3A binding observed in FIG. 3 in the presence of the pTM-NRP1peptide could be correlated to the inhibition of the NRP1oligomerization.

8) Functional Implication of pTM-NRP1-Dependent Inactivation of NRP1During Tumour Cell Migration

The rat C6 glioma cell line, which is a good model of human glioma (DAIand HOLLAND, Biochim. Biophys. Acta, vol. 1551, p: M19-27, 2001), hasbeen used to investigate how the blockade of NRP1 by using our peptidicstrategy (pTM-NRP1) may interfere with cell migration and dissemination.

C6 cells (ATCC CCL-107) were stained using PKH26 (Sigma). Cells wereincubated with peptides (pTM-NRP1 10⁻⁸M or mutated pTM-NRP1 10⁻⁸M) priorto injections for at least 2 h on ice in culture medium (composed of MEMwith 5000 u/ml penicillin, 5 mg/ml streptomycin, 200 mM L-glutamine and10% fetal calf serum). Injections of 10⁶ cells were performed using astereotaxic frame according to the following coordinates:antero-posterior, +1.6 mm relative to Bregma; L, +2 mm; H, +5 mmrelative to the cortical surface.

All injections were performed in the left striatum.

Following a survival period of 8 days, the animals (3 groups of 4 rats)were killed by a lethal intra-peritoneal injection of pentobarbitalbefore trans-cardiac perfusion with a pre-rinse of 100 ml PBS followedby 500 ml of 2% formaldehyde. The brains were post-fixed during 2 hoursat 4° C. and sagittal sections (70 μm) were prepared on a vibratome.

One group of sections were mounted in PBS-glycerol (v/v) for microscopicobservation, and another one was treated for immunostaining of CD34.Sections were first incubated in PBS containing 5% calf normal serum for15 minutes at room temperature to block non-specific binding sites. Asecond incubation was performed for 1 hour at room temperature and thenovernight at 4° C. with a mouse anti-CD34 (1:200). The sections werewashed six times during 5 minutes in PBS, and were then incubated with agoat anti-mouse antibody bound to Alexa-488 (1:500; INTERCHIM) for 3hours at room temperature. Sections were washed six times during 5minutes in PBS and were finally mounted in PBS-Glycerol (v/v) beforemicroscopic analysis

The FIG. 11 shows the mouse brain sections after the brain injection ofC6 cells with or without pTM-NRP1 or pTM-NRP1^(mut) peptides. Thepositions of the Tumour, cortex, striatum, corpus callosum (cc),hippocampus (Hp) and lateral ventricle (VL) are depicted onmicrophotographs.

The FIG. 12 shows the mouse brain sections after the brain injection ofC6 cells with or without pTM-NRP1 or pTM-NRP1^(mut) peptides after animmunostaining of CD34.

The results show that, in control conditions, tumours developed in thestriatum and reached the corpus callosum and the cortical plate (FIG.11, n=4). Strikingly, when cells were treated with pTM-NRP1 prior toinjection, we observed a strong reduction of the tumour size at 8 days(n=4). As expected, C6 cells treated with mutated pTM-NRP1 inducedtumours similar to those observed with non-treated cells (n=4). Thus,the addition of pTM-NRP1 inhibits the development of C6 glioma.

The results show also that the reduction of tumour size in the presenceof pTM-NRP1 was accompanied by a strong reduction of theimmunoreactivity for CD34, a marker of neoangiogenesis (FIG. 12). Thissuggested that pTM-NRP1 exerts its anti-tumour effect by blocking VEGFsignalling.

9) pTM-NRP1 can Antagonize VEGF Signalling In Vitro

NRP1 is a receptor of VEGF (NEUFELD et al., Adv. Exp. Med. Biol., vol.515, p: 81-90, 2002). We therefore verified that pTM-NRP1 can antagonizeVEGF signalling in C6 cells. To this end, C6 tumour cell aggregatesprepared as previously described (see BAGNARD et al., 1998; and NASARREet al., Neoplasia, vol. 7, p: 180-189, 2005) were grown in the 3D matrix(plasma clot) and treated with VEGF165.

The FIG. 13 shows representative tumour cell aggregates with or withoutthe addition of VEGF165 (50 ng/ml), pTM-NRP1 or pTMNRP1^(mut) (10⁻⁸ M).

The results show that the addition of 50 ng/ml VEGF165 induced C6 cellsmigration out of the aggregates and formation of migration chains (FIG.13). Strikingly, the addition of pTM-NRP1 suppressed VEGF165-dependentC6 cells migration. The addition of mutated pTM-NRP1 was not able toblock VEGF165-induced C6 cell migration. These results suggest thatpTM-NRP1 is able to block VEGF165 signalling in C6 cells therebyreducing tumour cells dissemination.

Finally, these results strongly suggest that that pTM-NRP1 can be usedto block NRP1 signalling in the context of tumorigenesis. This isrelated to the role of NRP1 during tumour cell migration and survivalthrough VEGF-dependent mechanisms. We propose that the blockade of NRP1using pTM-NRP1 has a therapeutic outcome for any tumour whose survival,growth and or dissemination requires a NRP1-dependent signallingcascade.

1. A peptidic antagonist of class III semaphorins/neuropilins complexesconsisting of: a transmembrane domain of a protein selected from thegroup consisting of neuropilin-1, neuropilin-2, plexin-A1, plexin-A2,plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrin beta 1 and integrin beta2, wherein the transmembrane domain is optionally fused to aheterologous sequence, or an amino acid sequence of less than 50 aminoacid long and more than 14 amino acid long having an identity of morethan 80% with a transmembrane domain of a protein selected from thegroup consisting of neuropilin-1, neuropilin-2, plexin-A1, plexin-A2,plexin-A3, plexin-A4, Nr-CAM, L1-CAM, integrin beta 1 and integrin beta2 and including at least one GxxxG motif, wherein the amino acidsequence of less than 50 amino acid long and more than 14 amino acidlong retains the biological properties of the transmembrane domain andwherein the amino acid sequence of less than 50 amino acid long and morethan 14 amino acid long is optionally fused to the heterologoussequence.
 2. The peptidic antagonist of claim 1, wherein the amino acidsequence of less than 50 amino acid long and more than 14 amino acidlong has an identity of more than 85% with the transmembrane domainamino acid sequence.
 3. The peptidic antagonist of claim 1, wherein theamino acid sequence of less than 50 amino acid long and more than 14amino acid long has an identity of more than 90% with the transmembranedomain amino acid sequence.
 4. The peptidic antagonist of claim 1,wherein the transmembrane domain has the amino acid sequence of thetransmembrane domain of human neuropilin-1 as set forth in SEQ ID NO: 1,of human neuropilin-2 as set forth in SEQ ID NO: 2, of human plexin A1as set forth in SEQ ID NO: 3, of human plexin A2 as set forth in SEQ IDNO: 11, of human plexin A3 as set forth in SEQ ID NO: 12, of humanplexin A4 as set forth in SEQ ID NO: 6, of human Nr-CAM as set forth inSEQ ID NO: 7, of human L1-CAM as set forth in SEQ ID NO: 13, of humanintegrin beta 1 as set forth in SEQ ID NO: 9 or of human integrin beta 2as set forth in SEQ ID NO:
 14. 5. The peptidic antagonist of claim 1,wherein the heterologous sequence allows at least one of: specificcellular location of the peptidic antagonist or improved purification ofthe peptidic antagonist.
 6. A nucleic acid encoding the peptidicantagonist according of claim
 1. 7. A vector comprising the nucleic acidof claim
 6. 8. A composition comprising the peptidic antagonist of claim1 and at least one pharmaceutically acceptable carrier.
 9. Thecomposition of claim 8, wherein the composition comprises aconcentration of more than 10⁻¹² M of the peptidic antagonist.
 10. Thecomposition of claim 8, wherein the composition comprises aconcentration of more than 10⁻¹¹ M of peptidic antagonist.
 11. Acomposition comprising the nucleic acid of claim 6 and at least onepharmaceutically acceptable carrier.
 12. A composition comprising thevector of claim 7 and at least one pharmaceutically acceptable carrier.13. A method for treatment of a subject suffering from a diseaseassociated with class III semaphorins/neuropilins complexes signaltransduction pathways, comprising administering to the subject apharmaceutical composition comprising a peptidic antagonist of class IIIsemaphorins/neuropilins complexes, the peptidic antagonist consisting ofa transmembrane domain of a protein selected from the group consistingof plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, and integrin beta 2,or consisting of a transmembrane domain of a protein selected from thegroup consisting of plexin-A2, plexin-A3, plexin-A4, Nr-CAM, L1-CAM, andintegrin beta 2, wherein the transmembrane domain is fused to aheterologous sequence, wherein the disease is selected from the groupconsisting of diseases associated with excessive angiogenesis andcancers.
 14. The method of claim 13, wherein administration of thepharmaceutical composition releases a concentration of the peptidicantagonist of more than 10⁻¹² M.
 15. The method of claim 13, wherein thetransmembrane domain has an amino acid sequence of the transmembranedomain of human plexin A2 as set forth in SEQ ID NO: 11, of human plexinA3 as set forth in SEQ ID NO: 12, of human plexin A4 as set forth in SEQID NO: 6, of human Nr-CAM as set forth in SEQ ID NO: 7, of human L1-CAMas set forth in SEQ ID NO: 13, or of human integrin beta 2 as set forthin SEQ ID NO:
 14. 16. The method of claim 13, wherein the heterologoussequence allows at least one of: specific cellular location of thepeptidic antagonist or improved purification yield of the peptidicantagonist.